Gene therapy is the purposeful delivery of genetic material to cells for the purpose of treating disease or biomedical investigation and research. Gene therapy includes the delivery of a polynucleotide to a cell to express an exogenous nucleotide sequence, to inhibit, eliminate, augment, or alter expression of an endogenous nucleotide sequence, or to produce a specific physiological characteristic not naturally associated with the cell. In some cases, the polynucleotide itself, when delivered to a cell, can alter expression of a gene in the cell. A basic challenge in gene therapy is to develop approaches for delivering genetic information to cells in vivo in a way that is efficient and safe. If genetic material are appropriately delivered they can potentially enhance a patient's health and, in some instances, lead to a cure. Delivery of genetic material to cells in vivo is also beneficial in basic research into gene function as well as for drug development and target validation for traditional small molecule drugs.
Skeletal muscle is an attractive target tissue for gene therapy interventions which aim to treat diseases such as muscular dystrophy or peripheral limb ischemia. Other inborn errors of metabolism and genetic muscle conditions, muscle diseases, muscle atrophy, muscle injury (including sports injuries) and secondary manifestations of muscular dystrophy are also candidates for treatment using gene therapy. In addition to muscle related diseases, other non-muscle conditions may also be treated through gene delivery to skeletal muscle. By delivering genetic material to skeletal muscle cells, muscle tissue could become a modified endocrine tissue. If the delivered gene encodes a protein that is secreted from the muscle cell, diseases such as hemophilia, diabetes, hypercholesterolemia, renal interstitial fibrosis, hypertension, dyslipoproteinemia, chronic renal fibrosis, liver cirrhosis, hyperglycemia, and atherosclerosis may be treated. Gene delivery to muscle cells may also be used to modulate or induce an immune reaction, to treat bone diseases or promote bone healing, or to treat growth plate injuries. While candidate genes have been identified that would likely be therapeutic, current delivery methods have associated problems.
It was first observed that injection of plasmid DNA directly into muscle in vivo enabled expression of foreign genes in the muscle (Wolff et al. 1990). More recently, intra-arterial delivery of polynucleotides to limb skeletal muscle has been shown to be effective (Liu et al. 1999, Lewis et al. 2002, Budker et al. 1996, McCaffrey et al. 2002, Zhang et al. 1999, Budker et al. 1998, Zhang et al. 2001, Liu et al. 2001, Hodges et al. 2003, Eastman et al. 2002). This method provided an improvement over direct muscular injection in affecting delivery of polynucleotides to muscle cells throughout a limb. Transfection efficiencies of >10% of myofibers in multiple muscle groups of the limb were obtained following a single injection into an arterial site (Budker et al. 1998, Zhang et al. 2001). While intra-arterial delivery of polynucleotides to limb skeletal muscle cells has proven to be effective, the procedure is not readily clinically viable. Arterial injections require invasive procedures to access the artery, making questionable whether repeat deliveries are clinically practical. Also, the large injection volumes and high injection rate needed for effective delivery are a cause of concern. Because of the presence of numerous valves in limb veins, it was believed that intravenous injection was not a viable option for delivering polynucleotides to limb muscle in vivo. Injection towards increased branching of the vein, as is done in arterial injection, would be blocked by these valves and would potentially damage the valves.
We now describe an effective in vivo delivery method that overcomes the obstacles presented by valves and uses limb veins for efficient, repeatable, and safe delivery of polynucleotides to skeletal myofibers throughout the limb muscles of mammals. The venous system is an attractive administration route, because like arteries, it is a direct conduit to multiple muscle groups of the limb. Unlike arteries, veins are much easier to access through the skin and there are less potential deleterious consequences relating to vessel damage during injection. In addition, a venous approach provides a more direct conduit to the post-capillary venules, which are more permeable to macromolecules than other parts of the microvasculature in muscle (Palade et al. 1978).